人G250真核表达载体的构建及稳定转染B16细胞系的建立

Construction of human G250 eukaryotic expression vector and establishment of stable B16 cell line transfection

目的构建人G250真核表达载体,建立稳定表达人G250的小鼠黑色素瘤细胞系。方法采用PCR方法扩增出人G250基因的cDNA编码区序列,利用DNA重组技术将其定向插入到真核表达载体pIRES-neo-sig中,并加入酶切位点和FLAG标签,得到重组表达质粒pIRES-neo-sig-FLAG-G250。利用阳离子脂质体介导法将其稳定转染入小鼠黑色素瘤B16细胞,经G418加压筛选出阳性克隆。RT-PCR及免疫荧光检测人G250在B16细胞中的表达。结果经限制性内切酶鉴定及序列分析,pIRES-neo-sig-FLAG-G250重组质粒构建正确,最终建立的表达人G250基因的B16细胞系阳性率接近100%。结论成功构建了真核表达载体pIRES-neo-sig-FLAG-G250,建立的稳定转染人G250小鼠黑色素瘤细胞系能够高效表达G250基因。该稳定转染细胞系的建立为G250在肿瘤免疫治疗中的应用奠定了研究基础。

Objective To construct the human G250 eukaryotic expression vector and establish the stable B16 cell line expressing human G250 in mice.Methods Human G250 cDNA was sequenced by PCR amplification and inserted into the eukaryotic expression vector pIRES-neo-sig with DNA recombinant techniques.Restriction enzyme position and FLAG tag were added into the vector to obtain the recombinant expression plasmid pIRES-neo-sig-FLAG-G250 which was transfected into B16 cells with Lipofectamine 2000.Positive clones were screened with G418.Expression of human G250 in B16 cells was detected by RT-PCR and immunofluorescence.Results Restriction enzyme and sequence analysis showed that the recombinant plasmid vector pIRES-neo-sig-FLAG-G250 was successfully constructed.The positive expression rate of human G250 in B16 cell line was nearly 100%.Conclusion A human G250 eukaryotic expression vector pIRES-neo-sig-FLAG-G250 can be constructed.Stable transfection of mouse B16 cell line can highly express G250,thus laying a foundation for the application of G250 in immunotherapy for tumors.